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RNA-dependent RNA polymerase (RdRp), (RDR), or RNA replicase, is an enzyme that catalyzes the replication of RNA from an RNA template. This is in contrast to a typical DNA-dependent RNA polymerase, which catalyzes the transcription of RNA from a DNA template.

RNA-dependent RNA polymerase (RdRp) is an essential protein encoded in the genomes of all RNA-containing viruses with no DNA stage that have sense negative RNA.[2][3] It catalyses synthesis of the RNA strand complementary to a given RNA template. The RNA replication process is a two-step mechanism. First, the initiation step of RNA synthesis begins at or near the 3' end of the RNA template by means of a primer-independent (de novo), or a primer-dependent mechanism that utilizes a viral protein genome-linked (VPg) primer. The de novo initiation consists in the addition of a nucleotide tri-phosphate (NTP) to the 3'-OH of the first initiating NTP. During the following so-called elongation phase, this nucleotidyl transfer reaction is repeated with subsequent NTPs to generate the complementary RNA product.[4]

Contents

History

Viral RdRPs were discovered in the early 1960s from studies on mengovirus and polio virus when it was observed that these viruses were not sensitive to actinomycin D, a drug that inhibits cellular DNA-directed RNA synthesis. This lack of sensitivity suggested that there is a virus-specific enzyme that could copy RNA from an RNA template and not from a DNA template.

The most famous example of RdRP is that of the polio virus. The viral genome is composed of RNA, which enters the cell through receptor-mediated endocytosis. From there, the RNA is able to act as a template for complementary RNA synthesis, immediately. The complementary strand is then, itself, able to act as a template for the production of new viral genomes that are further packaged and released from the cell ready to infect more host cells. The advantage of this method of replication is that there is no DNA stage; replication is quick and easy. The disadvantage is that there is no 'back-up' DNA copy.

Many eukaryotes also have RdRPs involved in an amplification of RNA interference. In them RdRP transcribes secondary- siRNAs, which in turn are bound by class 3 Argonauts (SAGO) to repress target RNA.[5] In fact these same RdRPs that are used in the defense mechanisms can be usurped by RNA viruses for their benefit.[citation needed]

RdRps are highly conserved throughout viruses and is even related to telomerase, though the reason for such high conservation in such diverse organisms is an ongoing question as of 2009.[6] The similarity has led to speculation that viral RdRps are ancestral to human telomerase.

Structure

All the RNA-directed RNA polymerases, and many DNA-directed polymerases, employ a fold whose organization has been likened to the shape of a right hand with three subdomains termed fingers, palm, and thumb.[7] Only the palm subdomain, composed of a four-stranded antiparallel beta-sheet with two alpha-helices, is well conserved among all of these enzymes. In RdRp, the palm subdomain comprises three well-conserved motifs (A, B, and C). Motif A (D-x(4,5)-D) and motif C (GDD) are spatially juxtaposed; the Asp residues of these motifs are implied in the binding of Mg2+ and/or Mn2+. The Asn residue of motif B is involved in selection of ribonucleoside triphosphates over dNTPs and, thus, determines whether RNA rather than DNA is synthesized.[8] The domain organization[9] and the 3D structure of the catalytic centre of a wide range of RdPps, even those with a low overall sequence homology, are conserved. The catalytic centre is formed by several motifs containing a number of conserved amino acid residues.

Classification

There are 4 superfamilies of viruses that cover all RNA-containing viruses with no DNA stage:

Flaviviruses produce a polyprotein from the ssRNA genome. The polyprotein is cleaved to a number of products, one of which is NS5. Recombinant dengue type 1 virus NS5 protein expressed in Escherichia coli exhibits RNA-dependent RNA polymerase activity. This RNA-directed RNA polymerase possesses a number of short regions and motifs homologous to other RNA-directed RNA polymerases.[10]

This tab holds the annotation information that is stored in the Pfam
database. As we move to using Wikipedia as our main source of annotation,
the contents of this tab will be gradually replaced by the Wikipedia
tab.

Flaviviruses produce a polyprotein from the ssRNA genome. This protein is also known as NS5. This RNA-directed RNA polymerase possesses a number of short regions and motifs homologous to other RNA-directed RNA polymerases [2].

External database links

RNA-directed RNA polymerase (RdRp) (EC) is an essential protein encoded in the genomes of all RNA containing viruses with no DNA stage [PUBMED:2759231, PUBMED:8709232]. It catalyses synthesis of the RNA strand complementary to a given RNA template, but the precise molecular mechanism remains unclear.
The postulated RNA replication process is a two-step mechanism. First, the initiation step of RNA synthesis begins at or near the 3' end of the RNA template by means of a primer-independent (de novo) mechanism. The de novo initiation consists in the addition of a nucleotide tri-phosphate (NTP) to the 3'-OH of the first initiating NTP. During the following so-called elongation phase, this nucleotidyl transfer reaction is repeated with subsequent NTPs to generate the complementary RNA product [PUBMED:11531403].

All the RNA-directed RNA polymerases, and many DNA-directed polymerases, employ a fold whose organisation has been likened to the shape of a right hand with three subdomains termed fingers, palm and thumb [PUBMED:9309225]. Only the catalytic palm subdomain, composed of a four-stranded antiparallel beta-sheet with two alpha-helices, is well conserved among all of these enzymes. In RdRp, the palm subdomain comprises three well conserved motifs (A, B and C). Motif A (D-x(4,5)-D) and motif C (GDD) are spatially juxtaposed; the Asp residues of these motifs are implied in the binding of Mg2+ and/or Mn2+. The Asn residue of motif B is involved in selection of ribonucleoside triphosphates over dNTPs and thus determines whether RNA is synthesised rather than DNA [PUBMED:10827187].
The domain organisation [PUBMED:9878607] and the 3D structure of the catalytic centre of a wide range of RdPp's, even those with a low overall sequence homology, are conserved. The catalytic centre is formed by several motifs containing a number of conserved amino acid residues.

There are 4 superfamilies of viruses that cover all RNA containing viruses with no DNA stage:

Flaviviruses produce a polyprotein from the ssRNA genome. The polyprotein is cleaved to a number of products one of which is NS5. Recombinant dengue type 1 virus NS5 protein expressed in Escherichia coli exhibits RNA-dependent RNA polymerase activity.
This RNA-directed RNA polymerase possesses a number of short
regions and motifs homologous to other RNA-directed RNA
polymerases [PUBMED:8607261].

Gene Ontology

The mapping between Pfam and Gene Ontology is provided by InterPro.
If you use this data please
cite InterPro.

Domain organisation

Below is a listing of the unique domain organisations or architectures in which
this domain is found.
More...

The graphic that is shown by default represents the longest sequence
with a given architecture. Each row contains the following information:

the number of sequences which exhibit this architecture

a textual description of the architecture, e.g. Gla, EGF x 2, Trypsin.
This example describes an architecture with one Gla
domain, followed by two consecutive EGF domains, and
finally a single Trypsin domain

a link to the page in the Pfam site showing information about the
sequence that the graphic describes

Note that you can see the family page for a particular domain by
clicking on the graphic. You can also choose to see all sequences which
have a given architecture by clicking on the Show link
in each row.

Finally, because some families can be found in a very large number of
architectures, we load only the first fifty architectures by default.
If you want to see more architectures, click the button at the bottom
of the page to load the next set.

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Pfam Clan

This family is a member of clan RdRP
(CL0027),
which has the following description:

This clan represents the replicative RNA dependent RNA polymerase. from a variety of RNA viruses [1].

Alignments

We store a range of different sequence alignments for families. As well
as the seed alignment from which the family is built, we provide the
full alignment, generated by searching the sequence database using the
family HMM. We also generate alignments using four
representative proteomes (RP) sets, the NCBI sequence database,
and our metagenomics sequence database.
More...

There are various ways to view or download the sequence alignments that
we store. We provide several sequence viewers and a plain-text
Stockholm-format file for download.

Alignment types

We make a range of alignments for each Pfam-A family:

seed

the curated alignment from which the HMM for the family is
built

full

the alignment generated by searching the sequence database
using the HMM

Viewing

a Java applet developed at the University of Dundee. You will
need Java installed
before running jalview

HTML

an HTML page showing the whole alignment.Please
note: full Pfam alignments can be very large. These
HTML views are extremely large and often cause problems for browsers.
Please use either jalview or the Pfam viewer if you have trouble
viewing the HTML version

PP/Heatmap

an HTML-based representation of the alignment, coloured according to
the posterior-probability (PP) values from the HMM. As for the standard HTML
view, heatmap alignments can also be very large and slow to render.

Pfam viewer

an HTML-based viewer that uses
DAS
to retrieve alignment fragments on request

Reformatting

You can download (or view in your browser) a text representation of a
Pfam alignment in various formats:

Selex

Stockholm

FASTA

MSF

You can also change the order in which sequences are listed in the
alignment, change how insertions are represented, alter the characters
that are used to represent gaps in sequences and, finally, choose
whether to download the alignment or to view it in your browser
directly.

Downloading

You may find that large alignments cause problems for the viewers and
the reformatting tool, so we also provide all alignments in Stockholm
format. You can download either the plain text alignment, or a gzipped
version of it.

View options

We make a range of alignments for each Pfam-A family. You can see a
description of each
above.
You can view these alignments in various ways but please note that some
types of alignment are never generated while others may not be available
for all families, most commonly because the alignments are too large to
handle.

Seed(7)

Full(5252)

Representative proteomes

NCBI(4283)

Meta(0)

RP15(0)

RP35(1)

RP55(1)

RP75(1)

Jalview

View

View

View

View

View

View

HTML

View

View

View

View

PP/heatmap

1

View

View

View

Pfam viewer

View

View

1Cannot generate PP/Heatmap alignments for seeds; no PP data available

Key: available,
not generated,
— not available.

Format an alignment

Seed(7)

Full(5252)

Representative proteomes

NCBI(4283)

Meta(0)

RP15(0)

RP35(1)

RP55(1)

RP75(1)

Alignment:

Format:

Order:

TreeAlphabetical

Sequence:

Inserts lower caseAll upper case

Gaps:

Download/view:

DownloadView

Download options

We make all of our alignments available in Stockholm format.
You can download them here as raw, plain text files or as
gzip-compressed files.

You can also
download a FASTA format file containing the
full-length sequences for all sequences in the full alignment.

External links

MyHits provides a
collection of tools to handle multiple sequence alignments. For example,
one can refine a seed alignment (sequence addition or removal,
re-alignment or manual edition) and then search databases for remote
homologs using HMMER3.

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HMM logo

HMM logos is one way of visualising profile HMMs. Logos provide a
quick overview of the properties of an HMM in a graphical form. You can
see a more detailed description of HMM logos and find out how you can
interpret them
here.
More...

If you find these logos useful in your own work, please consider citing
the following article:

Trees

This page displays the phylogenetic tree for this family's seed
alignment. We use
FastTree
to calculate neighbour join trees with a local bootstrap based on 100
resamples (shown next to the tree nodes). FastTree calculates
approximately-maximum-likelihood phylogenetic trees from our seed
alignment.

Curation and family details

This section shows the detailed information about the Pfam family. You
can see the definitions of many of the terms in this section in the
glossary and a fuller
explanation of the scoring system that we use in the
scores section of the
help pages.

Currently selected:

This visualisation provides a simple graphical representation of
the distribution of this family across species. You can find the
original interactive tree in the
adjacent tab.
More...

This chart is a modified "sunburst" visualisation of
the species tree for this family. It shows each node in the
tree as a separate arc, arranged radially with the superkingdoms
at the centre and the species arrayed around the outermost
ring.

How the sunburst is generated

The tree is built by considering the taxonomic lineage of each
sequence that has a match to this family. For each node in the
resulting tree, we draw an arc in the sunburst. The radius of
the arc, its distance from the root node at the centre of the
sunburst, shows the taxonomic level ("superkingdom",
"kingdom", etc). The length of the arc represents
either the number of sequences represented at a given level, or
the number of species that are found beneath the node in the
tree. The weighting scheme can be changed using the sunburst
controls.

In order to reduce the complexity of the representation, we
reduce the number of taxonomic levels that we show. We consider
only the following eight major taxonomic levels:

superkingdom

kingdom

phylum

class

order

family

genus

species

Colouring and labels

Segments of the tree are coloured approximately according to
their superkingdom. For example, archeal branches are coloured
with shades of orange, eukaryotes in shades of purple, etc. The
colour assignments are shown under the sunburst controls. Where
space allows, the name of the taxonomic level will be written on
the arc itself.

As you move your mouse across the sunburst, the current node
will be highlighted. In the top section of the controls panel we
show a summary of the lineage of the currently highlighed node.
If you pause over an arc, a tooltip will be shown, giving the
name of the taxonomic level in the title and a summary of the
number of sequences and species below that node in the tree.

Anomalies in the taxonomy tree

There are some situations that the sunburst tree cannot easily
handle and for which we have work-arounds in place.

Missing taxonomic levels

Some species in the taxonomic tree may not have one or more of
the main eight levels that we display. For example, Bos
taurus is not assigned an order in the NCBI taxonomic tree.
In such cases we mark the omitted level with, for example,
"No order", in both the tooltip and the lineage
summary.

Unmapped species names

The tree is built by looking at each sequence in the full
alignment for the family. We take the name of the species given
by UniProt and try to map that to the full taxonomic tree from
NCBI. In some cases, the name chosen by UniProt does not map to
any node in the NCBI tree, perhaps because the chosen name is
listed as a synonym or a misspelling in the NCBI taxonomy.

So that these nodes are not simply omitted from the sunburst
tree, we group them together in a separate branch (or segment of
the sunburst tree). Since we cannot determine the lineage for
these unmapped species, we show all levels between the
superkingdom and the species as "uncategorised".

Sub-species

Since we reduce the species tree to only the eight main
taxonomic levels, sequences that are mapped to the sub-species
level in the tree would not normally be shown. Rather than leave
out these species, we map them instead to their parent species.
So, for example, for sequences belonging to one of the
Vibrio cholerae sub-species in the NCBI taxonomy, we
show them instead as belonging to the species Vibrio
cholerae.

Too many species/sequences

For large species trees, you may see blank regions in the outer
layers of the sunburst. These occur when there are large numbers
of arcs to be drawn in a small space. If an arc is less than
approximately one pixel wide, it will not be drawn and the space
will be left blank. You may still be able to get some
information about the species in that region by moving your mouse
across the area, but since each arc will be very small, it will
be difficult to accurately locate a particular species.

Tree controls

Annotation

Download tree

Selected sequences

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View

graphically

as an
alignment

Download

sequence accessions

sequences in FASTA format

The tree shows the occurrence of this domain across different species.
More...

Species trees

We show the species tree in one of two ways. For smaller trees we try
to show an interactive representation, which allows you to select
specific nodes in the tree and view them as an alignment or as a set
of Pfam domain graphics.

Unfortunately we have found that there are problems viewing the
interactive tree when the it becomes larger than a certain limit.
Furthermore, we have found that Internet Explorer can become
unresponsive when viewing some trees, regardless of their size.
We therefore show a text representation of the species tree when the
size is above a certain limit or if you are using Internet Explorer
to view the site.

If you are using IE you can still load the interactive tree by
clicking the "Generate interactive tree" button, but please
be aware of the potential problems that the interactive species tree
can cause.

Interactive tree

For all of the domain matches in a full alignment, we count the
number that are found on all sequences in the alignment.
This total is shown in the purple box.

We also count the number of unique sequences on which each domain is
found, which is shown in green.
Note that a domain may appear multiple times on the
same sequence, leading to the difference between these two numbers.

Finally, we group sequences from the same organism according to the
NCBI
code that is assigned by
UniProt,
allowing us to count the number of distinct sequences on which the
domain is found. This value is shown in the
pink boxes.

We use the NCBI species tree to group organisms according to their
taxonomy and this forms the structure of the displayed tree.
Note that in some cases the trees are too large (have
too many nodes) to allow us to build an interactive tree, but in most
cases you can still view the tree in a plain text, non-interactive
representation. Those species which are represented in the seed
alignment for this domain are
highlighted.

You can use the tree controls to manipulate how the interactive tree
is displayed:

show/hide the summary boxes

highlight species that are represented in the seed alignment

expand/collapse the tree or expand it to a given depth

select a sub-tree or a set of species within the tree and view
them graphically or as an alignment

save a plain text representation of the tree

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Please note: for large trees this can take some time.
While the tree is loading, you can safely switch away from this
tab but if you browse away from the family page entirely, the tree
will not be loaded.

Structures

For those sequences which have a structure in the
Protein DataBank, we
use the mapping between UniProt, PDB and Pfam coordinate
systems from the PDBe group, to allow us to map
Pfam domains onto UniProt sequences and three-dimensional protein
structures. The table below
shows the structures on which the Flavi_NS5
domain has been found. There are 49
instances of this domain found in the PDB. Note that there may be
multiple copies of the domain in a single PDB structure, since many
structures contain multiple copies of the same protein seqence.